Project Summary Osteosarcoma (OSA), the most common primary malignant tumor of bone, has seen almost no improvement in patient overall survival rates since the 1980s. Approximately 30-40% of OSA patients develop tumor recurrence, with metastasis to the lung, occurring on average 1.6 years after diagnosis, accounting for 75-90% of recurrences. Once present, initially effective first-line therapies no longer provide clinical benefit in the metastatic setting, and only ~20% of these patients remain alive 4 years after recurrence. More concerning, there are no diagnostic methods which effectively identify those 30-40% of OSA patients at high risk for developing lung metastasis at time of initial diagnosis. While intensive investigation continues on intrinsic properties of tumor cells which confer their metastatic ability, emerging research has highlighted a critical role for non-malignant stromal cells of the host in extrinsic promotion of metastasis. Termed the pre-metastatic niche, studies in certain cancers have shown that tumor-derived soluble factors, including cytokines and exosomes, prime stromal cells of distant organs to generate microenvironments conducive to tumor cell outgrowth upon their subsequent arrival. Despite a significantly high and rapid rate of lung metastasis in OSA, little is known about the extrinsic, host-mediated mechanisms which govern this process. Interestingly, the group of pulmonary disorders termed interstitial lung disease (ILD) share significant overlap in their molecular pathogenesis of fibroblast-mediated lung remodeling with those of pre-metastatic niche formation. In both ILD and pre-metastatic niche formation, cytokines drive activation of a synthetic phenotype in resident lung fibroblasts, interestingly which is associated with transcriptomic changes in ILD patient-derived alveolar macrophages (AMs) predictive of this remodeling. Based on these observations, we hypothesize that OSA-derived soluble factors prime lung fibroblasts to foster metastasis, and that AMs can serve as effective sentinels of this priming via manifestation of a unique, metastasis-indicative molecular signature. To investigate this, we will utilize 3D organotypic co-culture of human OSA cells and primary lung fibroblasts as an in vitro model to recapitulate OSA lung micro-metastasis. We aim to compare differences in the secretome of paired high and low-metastatic phenotype human OSA cells cultured in standard 2D and 3D monoculture, versus co-culture with lung fibroblasts, via multiplex cytokine analysis, and quantitative mass spectrometry of exosomal protein cargo. Subsequently, we will determine the effects of these differentially generated tumor or tumor-fibroblast soluble factors on transcriptional changes in primary human AMs via RNA sequencing and differential gene expression profiling. Identification of the soluble factors and host cellular responses extrinsically driving OSA lung metastasis will provide the foundation for development of both biomarkers and novel therapies which identify and treat OSA patients at high-risk for lung metastasis.